Direct selection of transformants on acetate-containing media
A transformant selection system has been developed, particularly for a .beta.-lactam producing strain, more particularly for P. chrysogenum, by the complementation of a mutation of said .beta.-lactam producing strain by a homologous selection marker without interfering with .beta.-lactam biosynthesis. Particularly, in applying said transformant selection system a positive selection agent, for instance fluoroacetate is used for the isolation of fac mutants of said strain, particularly of said .beta.-lactam producing strain. Furthermore, a gene entitled facA has been isolated from P. chrysogenum.
Latest Gist-brocades B.V. Patents:
Claims
1. A method to select a transformed strain of the genus Penicillium, Aspergillus, or Acremonium which has been transformed with a first DNA of interest which method comprises:
- cotransforming a first mutant strain of said Penicillium, Aspergillus, or Acremonium which first mutant strain contains an inoperable facA gene and is unable to utilize acetate,
- with said first DNA of interest and with a selection marker DNA comprising an expression system effective in producing the acetyl CoA synthetase encoded by a fungal facA gene; and
- directly selecting first transformed strains for ability to grow on acetate-containing medium.
2. The method of claim 1 wherein at least some of said first transformed strains can be mutated to contain an inoperable facA gene and selected for said mutation to obtain a second mutant strain that contains an inoperable facA gene and is unable to utilize acetate.
3. The method of claim 1 wherein said cotransforming is conducted by supplying said first DNA of interest and said selectable marker DNA comprising said expression system on the same DNA molecule.
4. The method of claim 1 wherein the fungal acetyl CoA synthetase is homologous to the acetyl CoA synthetase of said first mutant strain.
5. The method of claim 1 wherein said first mutant strain is from the genus Penicillium.
6. The method of claim 1 where said first mutant strain is from the genus Aspergillus.
7. The method of claim 1 where said first mutant strain is from the genus Acremonium.
8. The method of claim 1 wherein said first mutant strain is selected from the group consisting of Penicillium chrysogenum, Aspergillus nidulans and Acremonium chrysogenum.
9. The method of claim 8 where said first mutant strain is P. chrysogenum.
10. The method of claim 8 where said first mutant strain is A. nidulans.
11. The method of claim 8 where said first mutant strain is A. chrysogenum.
12. The method of claim 4 wherein said first mutant strain is from the genus Penicillium.
13. The method of claim 4 where said first mutant strain is from the genus Aspergillus.
14. The method of claim 4 where said first mutant strain is from the genus Acremonium.
15. The method of claim 1 wherein said first mutant strain is a.beta.-lactam producing strain.
16. The method of claim 2 which further includes selecting said second mutant strain.
17. The method of claim 16 wherein said second mutant strain is selected by fluoroacetate resistance.
18. The method of claim 17 which further includes cotransforming said second mutant strain with a second DNA of interest and with a selection marker DNA comprising an expression system effective in producing the acetyl CoA synthetase encoded by a fungal facA gene.
19. The method of claim 18 which further includes selecting second transformants of said second mutant strain for ability to grow on acetate-containing medium.
20. A method to select a transformed strain of a.beta.-lactam producing fungus which has been transformed with a first DNA of interest which method comprises:
- cotransforming a first mutant strain of said.beta.-lactam producing fungus which first mutant strain contains an inoperable facA gene and is unable to utilize acetate,
- with said first DNA of interest and with a selection marker DNA comprising an expression system effective in producing the acetyl CoA synthetase encoded by a fungal facA gene; and
- directly selecting first transformed strains for ability to grow on acetate-containing medium.
21. The method of claim 20 wherein at least some of said first transformants can be mutated to contain an inoperable facA gene and selected for said mutation to obtain a second mutant strain that contains an inoperable facA gene and is unable to utilize acetate.
22. The method of claim 20 wherein said cotransforming is conducted by supplying said first DNA of interest and said selectable marker DNA comprising said expression system on the same DNA molecule.
23. The method of claim 20 wherein the acetyl CoA synthetase is homologous to the acetyl CoA synthetase of said first mutant strain.
24. The method of claim 21 which further includes selecting said second mutant strain.
25. The method of claim 24 wherein said second mutant strain is selected by fluoroacetate resistance.
26. The method of claim 24 which further includes cotransforming said second mutant strain with a second DNA of interest and with a selection marker DNA comprising an expression system effective in producing the acetyl CoA synthetase encoded by a fungal facA gene.
27. The method of claim 26 which further includes selecting second transformants of said second mutant strain for ability to grow on acetate-containing medium.
| 260762 | March 1986 | EPX |
| 215539 | March 1987 | EPX |
| 225078 | June 1987 | EPX |
| 235951 | September 1987 | EPX |
| 240250 | October 1987 | EPX |
- Diez et al. Mol. Gen Genet 218:572-576 (1989). Beri et al., Curr. Genet. (1987) 11:639-641. Kolar et al., Gene (1988) 62:127-134. Stahl et al., App. Microbiol. Biotechnol. (1987) 26:237-241. Picknett et al., FEMS Microbiol. Lett. (1989) 60:165-168. Whitehead et al., Mol. Gen. Genet. (1989) 216:408-411. Picknett et al., Curr. Genet. (1987) 12:449-455. Diez et al., Curr. Genet. (1987) 12:277-282. O'Sullivan et al., J. Gen. Microbiol. (1973) 76:65-75. Sandeman et al., Mol. Gen. Genet. (1989) 218:87-92. Thomas et al., Molec. Microbiol. (1988) 2:599-606. Connerton et al., Molec. Microbiol. (1990) 4:451-460. Hargreaves et al., J. Gen. Microbiol. (1989) 135:2675-2678. Apirion, Nature (1962) 195:959-961. Casselton et al., Mol. Gen. Genet. (1974) 132:255-264. Mellon et al., Int. Congr. Microbiol (1987) 14 Meet., 145.
Type: Grant
Filed: Jun 11, 1992
Date of Patent: Jul 20, 1999
Assignee: Gist-brocades B.V. (Ma Delft)
Inventors: Willem Van Hartingsveldt (Delft), Robert Franciscus Maria Van Gorcom (Delft), R. J. Gouka (The Hague), Roelof Ary Lans Bovenberg (Rotterdam)
Primary Examiner: John L. LeGuyader
Law Firm: Morrison & Foerster LLP
Application Number: 7/861,800
International Classification: C12Q 168; C12N 1580;
